Microsomal 3-hydroxy-3methlyglutaryl coenzyme A reductase (HMGR) of human liver was inactivated in vitro by ATP-Mg in a time dependent manner; this inactivation was mediated by reductase kinase (RK). Polyacrylamide gel electrophorisis of purified HMGR incubated with RK and (32P-ATP-Mg revealed that the 32P an HMGR enzyme activity were located in a single electrophoretic position. Dephosphorylation of 32P-HMGR was associated with loss of radioactivity and full restoration of enzyme activity. RK from human liver cytosol also exists in active and inactive forms. Active RK can be inactivated by incubation with phosphatase. Inactivated RK can be reactivated by incubation with ATP-Mg and a second kinase, reductase kinase kinase. These results represent the first demonstration of in vitro modulation of human liver HMGR by a bicycle cascade system involving reversible phosphorylation. We have also investigated the effect of cholesterol (CHL) and mevalonolactone (MVA) on the enzyme activity of rat liver HMGR. The effects of MVA and CHL are 2-fold. Short-term regulation involving inhibition of HMGR due to increase in phosphorylation; the long-term effect on HMGR is due to decrease in enzyme concentration.